The microenvironment of a cell comprises all the cues (stimuli) affecting the cell and includes attachment to neighboring cells, attachment to the structural molecules of the extracellular matrix (e.g., via integrins), molecules secreted by the cell itself or by other cells, nutrients, oxygen, mechanical stimuli (such as shear stress), and many others. As well as requiring information from each other, cells derive a vast wealth of information from their environments, including the material that surrounds and separates them within tissues, the extracellular matrix (ECM).
Within the biomedical field, areas such as tissue engineering, regenerative medicine, drug discovery, or cell biology would highly benefit from the development of 3D biomimetic environments that recreate the complexity of the natural extracellular matrix. The general strategy is usually to seed cells within a scaffold, a structural device that defines the geometry of the replacement tissue and provides environmental cues that promote tissue regeneration. Yet for commercial success tissue engineering products must be not only efficacious but also cost-effective, introducing a potential dichotomy between the need for sophistication and ease of production.
Due to this necessity, the last twenty years have seen a rapid increase in the development of two-dimensional (2D) surface patterning techniques that allow to control with high precision and reproducibility the positioning and presentation of different molecules on surfaces. These studies have had a huge impact in areas like tissue engineering, biosensors, and cell and molecular biology studies in general.
There are many fabrication processes available to generate functional patterns of molecules on 2D surfaces. Examples include soft lithography techniques, dip pen nano lithography, photolithography, nanoimprint lithography, or microfluidic devices (Chen C. S. et al. 1997. Science, 276 (5317): 1425-1428; Chiche A. et al. 2008. Soft Matter, 4(14):2360-2364). Nonetheless, 2D patterns are a weak representation of the real cell environment and therefore there is great need to create structures or scaffolds that exhibit similar spatial control of bioactive molecules as those of 2D surfaces but within 3D. The extracellular matrix found in living tissues is a complex 3D highly hydrated environment made from many elements such as soluble or surface bound molecules, proteins, enzymes, and physical cues like pores and topographies. The precise spatial location of these molecules plays a key role in the behaviour of cells, tissues, and ultimately organs (Perez-Castillejos R. 2010. Materials Today, 13(1-2): 32-41).
The field of fabricating 3D hydrogels with patterns is much less developed and only recently have researchers begun to explore different approaches. Emerging techniques include: bioprinting multilayered structures (Moon S. J. et al. 2010. Tissue Engineering: Part C Methods. 16: 157-166; Fernandez J. G. and Khademhosseini A. 2010. Advanced Materials 22: 2538-2541; Aubin H. et al. 2010. Biomaterials 31: 6941-6951), additive photopatterning (Liu Tsang V. et al. 2007. FASEB Journal, 21: 790-801), stereolithography (Khetan S. et al. 2010. Methods in Bioengineering. Eds.: M. L. Yarmush and R. S. Langer. Artech House Publishing), microfluidics (Wong A. P. et al. 2008. Biomaterials 29: 1853-1861) and sequential click reactions with photoaddition (DeForest C. A. et al. 2010. Chemistry of Materials, 22: 4783-4790; Johnson L. M. et al. 2010. ACS Appl Mater Interfaces, 2(7): 1963-1972; DeForest C. A. et al. 2009. Nature Materials, 8: 659-664). However, none of these techniques uses an electric field to move the molecules that are going to pattern the 3D hydrogel.
Albrecht D. R. et al. (Albrecht D. R. et al. 2005. Lab Chip, 5(1): 111-118) disclosed a method for encapsulating live cells in 3D-hydrogels using dielectrophoresis. However, the dielectrophoretic forces to locate cells are applied over a prepolymer suspension and said prepolymer suspension is then polymerized by exposure to UV light. The same dielectrophoretic forces have been reported to be useful to locate microgels containing encapsulating bioactive materials such as proteins or cells (Albrecht D. R. et al. 2007. Lab Chip, 7: 702-709). Nevertheless, this method requires the use of special chambers with a plurality of micropatterned surface electrodes to design the position of the molecules or cells in the hydrogel.
Techniques such as bioprinting, additive photopatterning, stereolithography and microfluidics have the disadvantage that they do not allow fabricating scaffolds with inhomogeneous concentrations of soluble molecules. On the other hand, sequential click reactions and dielectrophoresis have the disadvantage that the material is exposed to UV radiation. Moreover, some of them are expensive and it is necessary sophisticated equipment and chemical reactions to get a controllable pattern.
Furthermore, some approaches have been published that use 3D synthetic hydrogels to support cell growth (Cushing M. C. and Anseth K. S. 2007. Science 316: 1133-1134). Nevertheless, in tissue engineering the selection of a 3D scaffold for culturing cells is dictated by the capability of producing that scaffold in large quantities and better strategies must be developed for delivering endogenous factors to the right place within the gel.
Although a great number of techniques for patterning molecules in 3D hydrogels are known, there are some drawbacks which are still unsolved and limit their applications.
It is therefore necessary to develop further techniques for patterning molecules in 3D hydrogels to allow building more mimetic cell environments and which are capable of solving all or some of the above mentioned drawbacks related to the techniques of the state of the art.